Probiotic-Based Mineralized Living Materials to Produce Antimicrobial Yogurts

Abstract

Mineralization of living cells represents an evolutionary adaptation that enhances cellular resilience to physicochemical stress. Inspired by this strategy, we have here developed hybrid living materials (HLMs), incorporating probiotics into mineralized collagen 3D matrices, with the aim of protecting and promoting the successful oral delivery of the bacteria. Collagen fibrils are simultaneously self-assembled and mineralized in the presence of the probiotics (Lactobacillus acidophilus, La, was used as model), resulting in the integration of the probiotics into the hybrid matrix (i.e., bulk encapsulation). During this process, probiotics are also coated with a nanofilm of apatite mineral (single-cell encapsulation), which provides them with extra protection and reinforces their viability and activity. In fact, the resulting HLM is metabolically active, and maintain the capacity to ferment milk into yogurt with antibacterial activity against the two major foodborne pathogens Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa). Interestingly, the HLM provides probiotics an additional protection in the gastrointestinal environment (i.e., simulated gastric fluid), which is of special interest for healthcare materials for oral administration. The results pave the way for the creation of innovative healthcare materials with enhanced functionalities and the potential to produce probiotic foods with notable antimicrobial properties.

Keywords: antimicrobial; collagen; hybrid living materials; mineralization; probiotics; yogurt.

Figure 1

A) Schematic illustration of the experimental conditions for as‐synthesized materials. Solution A contains any or all of the following compounds: type‐I collagen monomers, La, and calcium ions. Solution B, also named as mineralizing solution, contains phosphate, carbonate, and citrate ions. The mixture of both solutions produces the corresponding HLMs. B–D) Representative SEM images of the HLMs: (B) probiotic incorporated in mineralized collagen (mCol@La), (C) probiotic incorporated in collagen (Col@La), and (D) mineralized probiotic (m@La).

Figure 2

Physicochemical characterization of mCol@La. A,B) TEM images and C,D) SAED pattern of the mineralized collagen fiber and mineral phase close to the collagen (black circles in Figure 2A 1A). E) HAADF‐STEM micrograph and EDS elemental maps of mCol@La showing the spatial distribution of N (blue), Ca (pink), and P (green) in the probiotics and collagen fiber. Scale bar = 2 µm. F) XRPD patterns and G) FTIR spectra of m@La, Col@La, mCol@La and of the abiotic components apatite nanocrystals (Ap) and mineralized collagen (mCol). The diffraction peaks of the Ap structures were confirmed and labeled according to the standard card of stoichiometric HA (JCPDS 9–432). H) Strain‐amplitude sweep test data at 1 Hz from Col@La and mCol@La. The linear viscoelastic region is maintained between 0.1–1% of shear strain, in which viscoelastic moduli (storage modulus G′ and loss modulus G′′) are constant. The pictures of I) mCol@La and J) Col@La illustrate the macroscopic appearance of the HLMs.

Figure 3

CLSM images of the HLMs and A) the corresponding three‐dimensional reconstruction and B) maximum intensity projections of mCol@La, C) m@La, and D) Col@La of 10 z‐sections with a 2‐microns z‐step. E) Pictures of the yogurt obtained with the HLMs and the corresponding controls. The diameter of the vials is 2 cm.

Figure 4

Total CFUs of La before (0 hour) and after incubation in gastric juice with La, m@La, Col@La, mCol@La, and the bacteria mixed with the abiotic components: Col+La, Col+La+mineral, and mCol+La. Statistical analysis was performed using one‐way ANOVA Bonferroni's post‐test. Asterisks and “ns” denote significant differences (p < 0.0015) and no significant differences, respectively.

Figure 5

Inhibitory activity of probiotic yogurts produced with the A) HLMs against B) Pa and C) Sa in TSA. D) Pathogens growth in contact with each of the yogurts. Bars of t₀ and control represent the initial loading in all the inhibition experiments and the growth of the pathogen in the absence of treatment, respectively. Statistical analysis was performed using one‐way ANOVA test. Asterisks indicate significant differences (p < 0.0001); ns denotes no significant differences.